HA-1-92, A new antifungal antibiotic produced by Streptomyces CDRIL-312: Fermentation,isolation, purification and biological activity

A new antifungal antibiotic, HA-1-92, was isolated from the biomass of Streptomyces CDRIL-312, by extracting in butanol and further purified by silica gel column chromatography followed by preparative TLC. The antibiotic is presumed to be an oxohexaene macrolide and showed promising antifungal activity against yeasts and filamentous fungi including human and plant pathogens. It was found to be less toxic in mice than known oxohexaenes.     

Overproduction of extracellular protease activity by Streptomyces C5-A13 in fed-batch fermentation

Summary Streptomyces C5-A13, a non-sporulating, pleiotropic mutant of the anthracycline-producing strain, Streptomyces C5, overproduces extracellular proteolytic activity against the substrate azocasein. This extracellular protease activity was produced primarily during the stationary phase. This appears to be an effect related to growth rate rather than to glucose repression, because only very high concentrations of glucose appear to inhibit protease synthesis. Production of extracellular protease activity was stimulated by the presence of carbonate anions in the medium. The optimal concentration of soluble carbonate was 60–80 mM and the stimulation by carbonate was shown not to be due to a pH effect. Approximately 3200–3500 units of extracellular azocaseinase activity were produced per millititre of culture broth using partially optimized fed-batch fermentation processes. This value represents about ninefold greater activity than produced under shake flask conditions.     

A novel extracellular subtilisin inhibitor produced by a Streptomyces sp

The amino acid composition and inhibitory properties of a protein (SI-1-72) isolated from the culture medium of a Streptomyces sp. have been investigated. SI-1-72 appears to be a monomer protein of molecular mass about 13,100 Da and amino acid composition which differs from that of the inhibitors of the Streptomyces subtilisin inhibitor (SSI) family. Furthermore, it was found to exhibit novel specificity: strong inhibitory effect against microbial alkaline proteinases, moderate effect towards chymotrypsin and elastase, and no inhibition of the other serine proteinases, as well as of the cysteine, aspartate and metallo-proteinases.     

Identification, isolation, and structural studies of extracellular polysaccharides produced by Caulobacter crescentus.

Caulobacters are adherent prosthecate bacteria that are members of bacterial biofouling communities in many environments. Investigation of the cell surface carbohydrates produced by two strains of the freshwater Caulobacter crescentus, CB2A and CB15A, revealed a hitherto undetected extracellular polysaccharide (EPS) or capsule. Isolation and characterization of the EPS fractions showed that each strain produced a unique neutral EPS which could not be readily removed from the cell surface by washing. Monosaccharide analysis showed that the main CB2A EPS contained D-glucose, D-gulose, and D-fucose in a ratio of 3:1:1, whereas the CB15A EPS fraction contained D-galactose, D-glucose, D-mannose, and D-fucose in approximately equal amounts. Methylation analysis of the main CB2A EPS showed the presence of terminal glucose and gulose groups, 3-linked fucosyl, and two 3,4-linked glucosyl units, thus confirming the pentasaccharide repeating unit indicated by 1H nuclear magnetic resonance analysis. Similar studies of the CB15A EPS revealed a tetrasaccharide repeating unit consisting of terminal galactose, 4-linked fucosyl, 3-linked glucosyl, and 3,4-linked mannosyl residues. EPS was not detectable by thin-section electron microscopy techniques, including some methods designed to preserve or enhance capsules, nor was the EPS readily detected on the cell surface by scanning electron microscopy when conventional fixation techniques were used; however, a structure consistent with EPS was revealed when samples were prepared by cryofixation and freeze-substitution methods.     

Optimization, purification, characterization and antioxidant activity of an extracellular polysaccharide produced by Paenibacillus polymyxa SQR-21

The optimization, purification and characterization of an extracellular polysaccharide (EPS) from a bacterium Paenibacillus polymyxa SQR-21 (SQR-21) were investigated. The results showed that SQR-21 produced one kind of EPS having molecular weight of 8.96 × 10⁵ Da. The EPS was comprised of mannose, galactose and glucose in a ratio of 1.23:1.14:1. The ratio of monosaccharides and glucuronic acid was 7.5:1. The preferable culture conditions for EPS production were pH 6.5, temperature 30 °C for 96 h with yeast extract and galactose as best N and C sources, respectively. The maximum EPS production (3.44 g L⁻¹) was achieved with galactose 48.5 g L⁻¹, Fe³⁺ 242 μM and Ca²⁺ 441 μM. In addition, the EPS showed good superoxide scavenging, flocculating and metal chelating activities while moderate inhibition of lipid peroxidation and reducing activities were determined. These results showed the great potential of EPS produced by SQR-21 to be used in industry in place of synthetic compounds.     

Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic fungi

Indigenous actinomycetes isolated from rhizosphere soils were assessed for in vitro antagonism against Colletotrichum gloeosporioides and Sclerotium rolfsii. A potent antagonist against both plant pathogenic fungi, designated SRA14, was selected and identified as Streptomyces hygroscopicus. The strain SRA14 highly produced extracellular chitinase and β-1,3-glucanase during the exponential and late exponential phases, respectively. Culture filtrates collected from the exponential and stationary phases inhibited the growth of both the fungi tested, indicating that growth suppression was due to extracellular antifungal metabolites present in culture filtrates. The percentage of growth inhibition by the stationary culture filtrate was significantly higher than that of exponential culture filtrate. Morphological changes such as hyphal swelling and abnormal shapes were observed in fungi grown on potato dextrose agar that contained the culture filtrates. However, the antifungal activity of exponential culture filtrates against both the experimental fungi was significantly reduced after boiling or treatment with proteinase K. There was no significant decrease in the percentage of fungal growth inhibition by the stationary culture filtrate that was treated as above. These data indicated that the antifungal potential of the exponential culture filtrate was mainly due to the presence of extracellular chitinase enzyme, whereas the antifungal activity of the stationary culture filtrate involved the action of unknown thermostable antifungal compound(s).     

Identification and characterization of an extracellular protease activity produced by the marine Vibriosp 60

Abstract The marine fish pathogen Vibrio sp. 60 has been used as a host for heterologous expression of the Escherichia coli heat-labile enterotoxin B-subunit and derivatives carrying a C-terminal extension. In this study, a chimeric enterotoxin B-subunit with an extension corresponding to the carboxy-terminal nine amino acids -Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-cooH from the small subunit of herpes simplex virus type 1-encoded ribonucleotide reductase, is shown to be proteolytically cleaved in the extracellular medium by a single protease that is secreted by the host strain. Such protease behaves as a typical metalloprotease, being inhibited by EDTA but not by a serine protease inhibitor. Purification and amino acid composition analysis of the two proteolysis products revealed a specific cleavage of the peptide bond between amino acids glycine and alanine of the nine amino acid extension with loss of activity. The above observation is relevant for the biotechnological exploitation of Vibrio sp. 60.     

Sch 42029, a naturally produced dopamine receptor ligand: Taxonomy,fermentation, isolation and structure

Summary A natural product, Sch 42029, isolated from the fermentation of anActinoplanes sp. (SCC 1971) was found to displace Sch 23390 from the dopamine-1 (D₁) receptor. The compound was isolated from the fermentation broth by adsorption of the filtrate on XAD-16 resin, elution with water-methanol, followed by purification by gel-permeation chromatography and HPLC. Using spectroscopic analysis, the structure was determined to be 2,5-dihydroxy acetanilide. The pure compound displaced Sch 23390, a D₁-selective ligand, at aK _i of 1.6 m and spiperone, a D₂-selective ligand, at aK _i of 200 m.     

AmfS, an extracellular peptidic morphogen in Streptomyces griseus

The amf gene cluster was previously identified as a regulator for the onset of aerial-mycelium formation in Streptomyces griseus. The nucleotide sequences of amf and its counterparts in other species revealed a conserved gene organization consisting of five open reading frames. A nonsense mutation in amfS, encoding a 43-amino-acid peptide, caused significant blocking of aerial-mycelium formation and streptomycin production, suggesting its role as a regulatory molecule. Extracellular-complementation tests for the aerial-mycelium-deficient phenotype of the amfS mutant demonstrated that AmfS was secreted by the wild-type strain. A null mutation in amfBA, encoding HlyB-like membrane translocators, abolished the extracellular AmfS activity without affecting the wild-type morphology, which suggests that AmfBA is involved not in production but in export of AmfS. A synthetic C-terminal octapeptide partially induced aerial-mycelium formation in the amfS mutant, which suggests that an AmfS derivative, but not AmfS itself, serves as an extracellular morphogen.     

Purification and properties of an extracellular pectin lyase produced by the strain of Penicillium oxalicum in solid-state fermentation

A pectin lyase (PNL, EC 4.2.2.10) produced extracellularly by the strain of Penicillium oxalicum in solid-state fermentation medium containing deoiled mandarin orange peel meal was purified to apparent homogeneity by a protocol that included ammonium sulfate precipitation, DEAE-Sephadex A-50 and Sephadex G-100 chromatography. The enzyme had molecular mass of 50 kD, as determined by SDS- PAGE and showed optimum pH and temperature at 8.0 and 50 degrees C respectively. It had an isoelectric point (pI) of 5.0 and showed a K(m) of 1.1 mg/ml of citrus pectin. The enzyme was strongly inhibited by Mo4+, Ag+ and Pb2+ and also by polyphenolic compounds, in particular tannic acid.     

Preparation,structural characterization and antioxidant activity of an extracellular polysaccharide produced by the fungus Oidiodendron truncatum GW

A water-soluble extracellular polysaccharide, designated Os2-1, was isolated from the fermented liquid of the fungus Oidiodendron truncatum GW using ethanol precipitation, anion-exchange and gel-permeation chromatography. Os2-1 was mainly composed of glucose with minor amounts of glucosamine, and its average molecular weight was about 9.6 kDa. On the basis of chemical and spectroscopic analyses, including one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR) spectroscopy, the backbone of Os2-1 was characterized to mainly consist of (1→6)-linked α-d-glucopyranose residues with minor amounts of (1→2)-linked α-d-glucopyranose residues. The antioxidant activity of Os2-1 was evaluated with the scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide radicals and inhibition effect of lipid peroxidation in vitro, and the results indicated that Os2-1 had good antioxidant activity, especially scavenging ability on DPPH and superoxide radicals. The investigation demonstrated that Os2-1was an extracellular polysaccharide differing from previously described extracellular polysaccharides, and could be a potential antioxidant.     

Characterization of an extracellular glycoprotein inhibitor of trypsin (TI-23) produced by Streptomyces sp. 23

Summary The production of an extracellular trypsin inhibitor, TI-23, was found to parallel the growth of Streptomyces sp. 23 at different cultivation temperatures, reaching a maximum level at late exponential phase. Although the different temperatures (18°, 28° and 37°C) did not greatly affect the growth of the microorganism, they proved to be an important factor for extracellular inhibitory activity. Maximum specific rates of both cell growth and production of the inhibitor were recorded during the cultivation of Streptomyces sp. 23 at 37°C. TI-23 proved to be a monomeric glycoprotein containing 17% carbohydrate and differing in amino acid composition from the known extracellular proteinase inhibitors of streptomycetes. The molecular mass of the inhibitor was estimated to be about 13 kDa and the isoelectric point 4.3. The inhibition spectrum of TI-23 included trypsin as well as some microbial alkaline proteinases.     

Antifungal activity of extracellular metabolites produced by Sporothrix flocculosa

The antifungal properties of extracellular compounds produced by the epiphytic fungus Sporothrix flocculosa were bioassayed against phytopathogenie fungi on the basis of inhibition of spore germination, and mycelial growth and induction of cellular leakage. Following incubation in stationary culture, S. flocculosa released antifungal metabolites into the culture medium which were extractable with méthylene chloride. When separated by thin layer chromatography, extracted metabolites yielded a compound(s) at R_f0.65 which inhibited development of Cladosporium cucumerinum and several other phytopathogenic fungi. Treatment of Botrytis cinerea and Fusarium oxysporum f.sp. radicis‐lycopersici (FORL) with the same compound(s) greatly reduced spore germination and biomass growth of both fungi. Additionally, both B. cinerea and FORL leaked electrolytes and proteins when grown in presence of the metabolites. Observations under electron microscopy revealed that FORL reacted to the presence of S. flocculosa metabolites by retraction of the plasmalemma and rapid disintegration of the cytoplasm. These reactions were similar to the ones induced by conidia of S. flocculosa when applied on powdery mildew fungi. These results provide strong evidence of the production of antifungal compounds in vivo and of their role in the antagonistic properties of S. flocculosa.     

CP-60,993, a new dianemycin-like ionophore produced byStreptomyces hygroscopicus ATCC 39305: fermentation,isolation and characterization

Summary CP-60,993, 19-epi-dianemycin, is a novel polycyclic ether antibiotic produced byStreptomyces hygroscopicus ATCC 39305. Fermentation recovery, purification and crystallization were achieved using standard procedures. CP-60,993 was characterized as a monocarboxylic acid. Elemental analysis suggested a molecular formula of C₄₇H₇₈O₁₄ for the free acid and C₄₇H₇₇O₁₄ Na for the sodium salt. Crystalline form CP-60,993 sodium salt shows the following properties: m.p. 193205°C, E _{1 cm} ^1% =157 at 232 nm, [] _D ^25°C +11.0 (c 1, methanol). The structure, determined by MS, PMR and CMR, differs from dianemycin only in the stereochemistry at position 19. This was confirmed by X-ray crystallography carried out on the rubidium salt of CP-60,993. It exhibited activity in vitro against Gram-positive and anaerobic bacteria, efficacy againstEimeria coccidia in vivo in poultry, and stimulation in vitro of rumen propionic acid production.     

Structure of heliomycin produced by Streptomyces heliomycini and antibiotic 11-98 produced by Streptomyces olivocinereus

The data on UV, 1H NMR and mass spectroscopy confirmed that heliomycin produced by S. heliomycini and antibiotic 11-98 produced by S. olivocinereus were identical with resistomycin. The major minor component produced by S. heliomycini was shown to be resistoflavin which was also confirmed by physicochemical methods.     

Studies of the extracellular proteolytic activity produced by Propionibacterium acnes

Of several commercial media tested, trypticase soya both containing 0.4% (w/v) D-sorbitol was superior as a growth medium for the production of extracellular proteinase by Propionibacterium acnes (strain P-37). Extracellular proteinase, production of which was shown to be growth-associated by both batch and continuous culture studies, was partially purified by 70% (NH4)2SO4 saturation. Sephadex G-75 chromatography and ion exchange on DEAE-Sephadex A-50. It was shown to be a heterogeneous mixture of at least three molecular species of enzyme. Proteinase I was inhibited by EDTA (10(-3) mol/l) and PMSF (5 millimol/l) and stimulated by CaCl2 (190% at 10(-3) mol/l). It had a molecular weight of 20 to 30000 and a broad pH optimum from 6.5 to 7.5. Proteinase II was an alkaline proteinase with a molecular weight of 30 to 40000 which was not significantly inhibited by EDTA (10(-2) mol/l) nor stimulated by CaCl2. Proteinase III represented a minor proportion of the recovered proteolytic activity, had a molecular weight of 20 to 30000 and was most active in the alkaline pH range. This enzyme was inhibited by EDTA (10(-4) mol/l) and PMSF (5 millimol/l), and stimulated by CaCl2 (250% at 10(-2) mol/l).     

Studies of the extracellular proteolytic activity produced by Propionibacterium acnes

Of several commercial media tested, trypticase soya broth containing 0.4% (w/v) D-sorbitol was superior as a growth medium for the production of extracellular proteinase by Propionibacterium acnes (strain P-37). Extracellular proteinase, production of which was shown to be growth-associated by both batch and continuous culture studies, was partially purified by 70% (NH₄)₂SO₄ saturation, Sephadex G-75 chromatography and ion exchange on DEAE-Sephadex A-50. It was shown to be a heterogeneous mixture of at least three molecular species of enzyme. Proteinase I was inhibited by EDTA (10^-3 mol/l) and PMSF (5 millimol/l) and stimulated by CaCl₂ (190% at 10^-3 mol/l). It had a molecular weight of 20 to 30000 and a broad pH optimum from 6.5 to 7.5. Proteinase II was an alkaline proteinase with a molecular weight of 30 to 40000 which was not significantly inhibited by EDTA (10^-2 mol/l) nor stimulated by CaCl₂. Proteinase III represented a minor proportion of the recovered proteolytic activity, had a molecular weight of 20 to 30000 and was most active in the alkaline pH range. This enzyme was inhibited by EDTA (10^-4 mol/l) and PMSF (5 millimol/l), and stimulated by CaCl₂ (250% at 10^-2 mol/l).